Balkan Journal of Medical Genetics

VALUE OF OPTICAL GENOME MAPPING (OGM) FOR DIAGNOSTICS OF RARE DISEASES: A FAMILY CASE REPORT
Kovanda A1,2, Miljanović O3, Lovrečić L1,2, Maver A1,2, Hodžić A1,2, Peterlin B1,2,*
*Corresponding Author: *Corresponding Author: Prof. Borut Peterlin, Clinical Institute of Genomic Medicine, University Medical Centre Ljubljana, Šlajmerjeva 4, 1000 Ljubljana, Slovenia. borut.peterlin@kclj.si
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RESULTS

Clinical characteristics Characteristics of two male siblings (proband 1 and proband 2) with an overlapping clinical presentation of thrombocytopenia, sacrococcygeal teratoma, hydrone- phrosis/reflux vesicoureteral and obesity, are shown in Table 1. Both parents and three sisters of the probands were healthy and without any of the clinical signs and symptoms shown in the probands, except for a few asymptomatic episodes of borderline platelet values in the mother. Karyotype analyses Normal male karyotypes were detected for both male siblings (Proband 1: 46, XY; Proband 2: 46, XY), with no clonal abnormalities (30 metaphases), at the stated band level of resolution. Karyotypes of the parents were also normal (Mother: 46, XX; Father: 46, XY). Microarray analyses Microarray analyses of the proband 1 showed an interstitial single copy gain of 18q12.2 region, approxi- mately 459,7 kb in size in a male profile: arr[GRCh38] 18q12.2(38880911_39340584)×3 (arr[GRCh37] 18q12.2(36460875_36920548)×3). The identified dupli- cation did not overlap with any known disease-causing genes and was not present in the databases containing vari- ants from healthy individuals (DGV), nor in the medical literature or databases ClinGen, ClinVar, or DECIPHER. Due to its size and rarity, the copy number gain was inter- preted as a variant of unknown significance, and segrega- tion analysis using arrays was recommended. Segregation testing using microarrays in the mother and father of the proband showed the presence of the same 18q12.2 copy number gain in the mother of the proband. As the molecular karyotyping showed the presence of the same interstitial duplication of the 18q12.2 region in the proband and the mother; arr[GRCh38] 18q12.2(38880911_39340584)×3m at, and the duplication did not affect clinically important genes, it was interpreted as a likely benign genomic variant, and so clinical testing was continued to determine the cause of the clinical presentation in the proband and his brother. Exome sequencing Exome sequencing was initially performed for pro- band 1, as previously described13,14. The original gene panel included >100 genes associated with thrombocytopenia and hereditary thrombocytopenia including Wiskott-Al- drich syndrome. The analysis did not identify any variants that could explain the phenotype and therefore a reinter- pretation of the exome sequencing data of proband 1 was performed with an expansion to genes associated with the additional phenotypes observed (Table 1). Despite adding over 1500 genes to the analysis, no causative variants were identified. Finally, exome sequencing in trio setup was performed for proband 2 and both parents with updated gene panels. Despite including >2000 genes associated with the clinical phenotypes, no conclusively causative SNV variants or small indels could be identified. While the duplication observed on the microarray analysis was appar- ent from the coverage, no breakpoints could be identified by exome sequencing. The full list of genes included in the exome sequencing analysis is available in the Supplement. Optical genome mapping Optical genome mapping showed a translocation between chromosomes X and 18, accompanied by a duplication of the 18q12.2 region, of maternal origin in both probands; ogm[GRCh38] t(X;18)(q27.1;q12.2)(14 0408784~140427850;38878133~39396298)mat,dup(18) (18q12.2)(38927193_39426970)mat. The translocation breakpoints and the associated duplication of the 18q12.2 region did not overlap any clinically significant genes and are unlikely to be visible using classic karyotyping methods. The accompanying 18q12.2 duplication was approximately 499,7 kb in size, and was consistent with the previously observed duplication in the proband 1, 2 and their mother using microarray analysis: arr[GRCh38] 18q12.2(38880911_39340584)×3mat (Figure 1). Interpretation and segregation analysis The translocation and accompanying duplication of maternal origin do not directly affect genes known to cause disease in humans. However, several genetic mechanisms are known to influence the expression of nearby genes by influencing regulatory regions or by topological means, some promoting and some inhibiting expression 19–22. Therefore, as described previously, we used the UCSC Genome Browser Viewer to visualize our region of interest in the context of neighboring genomic regions 12,23, how- ever no obvious regulatory regions explaining the pheno- type could be identified as being affected by the detected translocation and accompanying duplication. However, literature search revealed that the region of chromosome 18 involved in the rearrangement has previously been described in the context of germinal translocation t(11;18) (q22.1;q12.2), (ClinVar ID: 599287), where the transloca- tion carriers also had age-dependent hypertension linked to 11q22.1, as well as obesity 24. Additionally, somatic translocations between chromosome X and chromosome 18 involving different breakpoints have been previously described in synovial sarcomas (t(X;18)(p11.2;q11.2)), including in a rare case with submandibular presentation 25,26, however, the exact breakpoints of the critical region of 18q11.2 do not correspond to those identified in our patients. Therefore, because of the lack of direct evidence of pathogenicity, but because of the clinical match of the probands, the involvement of the chromosome X in males and a female with a very slight phenotype of transient thrombocytopenia, and the size of regions possibly affected indirectly, the translocation was classified as a variant of unknown clinical significance. When we are unable to provide final conclusions, extensive segregation may prove beneficial to clarifying the classification of the vari- ant, as recently described in case of a PLP1 duplication by our group12. In case of variants involving chromosome X in males, testing additional male family members may provide additional information helpful to clinical clari- fication, which is why we expanded the segregation to include healthy brothers of the carrier mother. The results of the segregation analysis are shown in Figure 2. None of the four maternal uncles were carriers of this rare fa- milial translocation, that remains a variant of unknown significance. Limitations OGM requires a special isolation/extraction step, producing ultra-long/high molecular weight DNA (hm- wDNA) molecules, that are typically in the 200 kilobases (kb) to megabase (Mb) range, in contrast to typical DNA isolation protocols where the resulting DNA is usually up to 20 kb in size. Therefore, archival DNA samples cannot be used for OGM, and fresh extraction is needed. After extraction, DNA is labeled across specific motifs using the DLE-1 enzyme, while the backbone DNA is also labeled using a special stain. The current technical limitations of OGM concern the size of DNA required, DLE-1 labeling limitations, and interpretation challenges. As large DNA molecules are needed for this method, it currently cannot be performed from archived DNA or FFPE, and therefore fresh samples are needed. Furthermore, the method cannot detect SV within regions that do not contain the DLE-1 labeling motif, such as Robertsonian translocations. Simi- larly, regions spanning segmental duplications, e.g. on the chrY chromosome can result in several alternative assem- blies. The interpretation of genomic variants in terms of pathogenicity is currently based on recommendations from ACMG and the joint consensus of the American College of Medical Genetics and Genomics (ACMG) and the Clinical Genome Resource (ClinGen)15,27. However, many of the different SVs detected by the OGM method, for example, balanced translocations, inversions, etc. lack clear guide- lines for classification, and so the interpretation of these SVs needs to be carefully considered on a case-by-case basis. The limited size of known normal OGM genetic variation at the moment means, that many identified vari- ants remain variants of unknown significance. Finally, because of its novelty, there is a need to establish a larger database of normal human genomic variation detected us-significance, highlighting the complexity of diagnostic results in rare disease cases as well as the remaining limita- tions of this technology. Hopefully, the future increase in healthy control population OGM variants and the estab- lishment of official guidelines on the clinical interpretation of OGM variants will resolve many current interpretation challenges.

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